1. NCBI Gene #: 2772192 2. Protein ID (NP or XP #) or Wolbachia#: YP_001316.1 3. Organism: Leptospira interrogans serovar Copenhageni; Strain: Fiocruz L1-130 4. Etiologic Risk Group: Risk Group 2 and Risk Group 3 (in Belgium) 5. Background/Disease Information: Leptospira interrogans is a a spirochete bacterium that can infect a variety of hosts, including dogs, rodents, and humans. It is spread through contact with the urine of an infected host. The contact can be direct or indirect, as through a standing body of water. Once contact has been made, the bacterium will enter the bloodstream and infected many different cells and organs, a process thought to be a result from Leptospira's ability to attach to multiple receptors on the surface of a host's cell. It is commonly referred to as rat catcher's yellow as the host's body often turns yellow from jaundice during the course of the infection.
The organs most infected are the kidney and the liver, which can cause complications like renal failure, Weil's Syndrome, and kidney infection. During the first phase of infection, the host experiences vague symptoms like headache, muscle pain, fever, and nausea. It is only during the second infectious phase that the life-threatening symptoms, such as meningitis, fever, and organ failure, appear. When the body of the host starts to shut down, the bacterium will leave its host through the urine, after which it can survive in water for up to three months. Leptospira most often infects people that work with animals or work outdoors, especially in tropical climates, such as farmers, fish workers, and mine workers. Symptoms can last for a few days to a few weeks or even longer, although an early dose of doxycycline or penicillin will cure the illness. 6. Link to TDR Targets page (if present): No TDR Targets Page present 7. Link to Gene Database page: http://www.ncbi.nlm.nih.gov/gene/2772192 8.Essentiality of this protein: This protein is essential to the function of the Leptospira organism because it is needed for the bacterium to interact with the extracellular matrix of the host cell, as well as modulating fibronectin binding. 9.Complex of proteins?: No 10.Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): http://www.ncbi.nlm.nih.gov/pubmed/12138134
Lipl32 has been targeted in Leptospira shermani, another pathogenic strain of Leptospira in mice, and targeted this protein reduced the nuclear binding ability of the bacterium. 11.EC#: 3.4.24.B14 12. Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.4.24.B14 13. Show screenshot of BRENDA enzyme mechanism schematic
Figure 1. Image of the reaction mechanism for the BRENDA enzyme EC 3.4.24.B14 - neprilysin-2.
14. Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): 14a.link to Sigma (or other company) page for assay (see Sigma links below) http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/peptidase.pdf 14b. links to assay reagents (substrates) pages. http://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=15844975 15.List cost and quantity of substrate reagents, supplier, and catalog # 15a.Cost: 421 USD 15b. Quantity: 1 15c.Supplier: SIGMA-ALDRICH 15d. Catalog #: D4943 16. Structure Available: PDB #2WFK 17. Current Inhibitors: None 18. Expression Information (has it been expressed in bacterial cells): It has been expressed in Liptosprira cells. 19. Purification Method: X-Ray Diffraction 20. Image of protein (PyMol with features delineated and shown separately):
Figure 2. PyMol image of the calcium-bound protein Lipl32. The protein chains are colored from the N- to the C-terminal using rainbows.
21. Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
KQAIAAEESLKKAASDATK 22. length of your protein in Amino Acids: 259 Amino Acids 23. Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 28522.4 kDa 24. Molar Extinction coefficient of your protein at 280 nm wavelength: 38390 25. TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Figure 3. TMPRED graph for the amino acid sequence of the calcium-bound protein Lipl32.
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
Target: Calcium Bound LIPL32 (pathogenic Leptospira)
1. NCBI Gene #: 27721922. Protein ID (NP or XP #) or Wolbachia#: YP_001316.1
3. Organism: Leptospira interrogans serovar Copenhageni; Strain: Fiocruz L1-130
4. Etiologic Risk Group: Risk Group 2 and Risk Group 3 (in Belgium)
5. Background/Disease Information:
Leptospira interrogans is a a spirochete bacterium that can infect a variety of hosts, including dogs, rodents, and humans. It is spread through contact with the urine of an infected host. The contact can be direct or indirect, as through a standing body of water. Once contact has been made, the bacterium will enter the bloodstream and infected many different cells and organs, a process thought to be a result from Leptospira's ability to attach to multiple receptors on the surface of a host's cell. It is commonly referred to as rat catcher's yellow as the host's body often turns yellow from jaundice during the course of the infection.
The organs most infected are the kidney and the liver, which can cause complications like renal failure, Weil's Syndrome, and kidney infection. During the first phase of infection, the host experiences vague symptoms like headache, muscle pain, fever, and nausea. It is only during the second infectious phase that the life-threatening symptoms, such as meningitis, fever, and organ failure, appear. When the body of the host starts to shut down, the bacterium will leave its host through the urine, after which it can survive in water for up to three months. Leptospira most often infects people that work with animals or work outdoors, especially in tropical climates, such as farmers, fish workers, and mine workers. Symptoms can last for a few days to a few weeks or even longer, although an early dose of doxycycline or penicillin will cure the illness.
6. Link to TDR Targets page (if present): No TDR Targets Page present
7. Link to Gene Database page:
http://www.ncbi.nlm.nih.gov/gene/2772192
8.Essentiality of this protein: This protein is essential to the function of the Leptospira organism because it is needed for the bacterium to interact with the extracellular matrix of the host cell, as well as modulating fibronectin binding.
9.Complex of proteins?: No
10.Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
http://www.ncbi.nlm.nih.gov/pubmed/12138134
Lipl32 has been targeted in Leptospira shermani, another pathogenic strain of Leptospira in mice, and targeted this protein reduced the nuclear binding ability of the bacterium.
11.EC#: 3.4.24.B14
12. Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.4.24.B14
13. Show screenshot of BRENDA enzyme mechanism schematic
14. Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
14a.link to Sigma (or other company) page for assay (see Sigma links below)
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Enzyme_Assay/peptidase.pdf
14b. links to assay reagents (substrates) pages.
http://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=15844975
15.List cost and quantity of substrate reagents, supplier, and catalog #
15a.Cost: 421 USD
15b. Quantity: 1
15c.Supplier: SIGMA-ALDRICH
15d. Catalog #: D4943
16. Structure Available: PDB #2WFK
17. Current Inhibitors: None
18. Expression Information (has it been expressed in bacterial cells):
It has been expressed in Liptosprira cells.
19. Purification Method: X-Ray Diffraction
20. Image of protein (PyMol with features delineated and shown separately):
21. Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
>2WFK:D|PDBID|CHAIN|SEQUENCE
DRWIRPRGAFGGLPSLKSSFVLSESTVPGTNETVKTFLPYGSVINYYGYVKPGQAPDGLVDGNKKAYYLYVWIPAVIAEM
GVRMISPTGEIGEPGDGDLVSDAFKAATPEEKSMPHWFDTWIRVERMSAIMPDQIAKAAKAKPVQKLDDDDDGDDTYKEE
RHNKYNSLTRIKIPNPPKSFDDLKNIDTKKLLVRGLYRISFTTYKPGEVKGSFVASVGLLFPPGIPGVSPLIHSNPEELQ
KQAIAAEESLKKAASDATK
22. length of your protein in Amino Acids: 259 Amino Acids
23. Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 28522.4 kDa
24. Molar Extinction coefficient of your protein at 280 nm wavelength: 38390
25. TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
26. CDS Gene Sequence (paste as text only):
mkklsilais valfasitac gafgglpslk ssfvlsedti pgtnetvktl lpygsvinyy
gyvkpgqapd glvdgnkkay ylyvwipavi aemgvrmisp tgeigepgdg dlvsdafkaa
tpeeksmphw fdtwirverm saimpdqiak aakakpvqkl dddddgddty keerhnkyns
ltrikipnpp ksfddlknid tkkllvrgly risfttykpg evkgsfvasv gllfppgipg
vsplihsnpe elqkqaiaae eslkkaasda tk
27. GC% Content for gene: 8.0882352941176
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**